Intracellular antibodies or "intrabodies" exploit the specificity and diversity of immunoglobulins to knockdown the function of intracellular proteins through antibody expression inside cells. In addition to their specificity, intrabodies offer several advantages over the gene deletion and RNAi-based strategies that are commonly used to eliminate protein function. For example, intrabodies can be engineered to block only certain domains of a protein, allowing the ability to target specific functions of a protein for knockdown. The ability to decouple functions of a protein target could be especially useful when the protein targeted for knockdown has an essential function. In addition, intrabodies can be multivalent, so a single intrabody could be engineered to simultaneously eliminate the function of two or more intracellular targets. Although intrabodies show great promise, generating antibodies for specific intracellular proteins is generally more time-consuming and labor- intensive than gene-based knockout approaches. The reducing environment of the cell cytoplasm and nucleus also presents a technical challenge, since it prevents the formation of intrachain disulfide bonds that are essential for the folding of nearly all antibodies. The ultimate objective for this work is to overcome these limitations and develop a proteome-wide set of intrabodies that can be used to study and modulate the activity of intracellular proteins. This research training program works toward the long-term objective by engineering functional intrabodies for defined protein targets in human cancer cells. The specific aims of this research training program are to (1) isolate novel intrabodies against targets involved in MAPK signaling and differentiation and (2) validate the engineered intrabodies against their targets in HL-60 cells. Intrabodies will be engineered in Escherichia coli using an inner membrane antibody display platform that incorporates the intracellular folding quality control mechanism of the bacterial twin-arginine translocation (Tat) pathway. The intrabodies will target CD38 and c-Cbl, proteins that have critical roles in MAPK signaling. Following screening of intrabodies displayed on the E. coli inner membrane, the intrabodies will be expressed in HL-60 cells to validate their functionality and determine their effect on HL-60 differentiation. This research will result in an intrabody-mediated protein knockdown strategy that will provide valuable information about the molecular mechanisms involved in HL-60 differentiation, and the information gained could be used to determine whether specific intracellular proteins are viable targets for therapeutic intervention. PUBLIC HEALTH RELEVANCE: Because of their diversity and specificity, intracellular antibodies (intrabodies) have the potential to alter a vast array of biological processes by functioning inside of living cells. Intrabodies that target signaling and differentiation targets in human leukemia cells will be engineered and validated. The project will develop strategies that can be applied to other cellular models to determine whether specific intracellular proteins are viable targets for therapeutic intervention.